choice of stable mammalian expression/knock-in

[patrickzhang76 from gmail.com]

Hi there. I want to "knock-in" a gene that is not expressed in a
mammalian cell line. I want to make a stable line. In the old days it
is not a problem since there are not many choices out there. However,
with new lentiviral technology, I need to make a choice between
transfection vs. viral transduction.

People told me the traditional "antibiotic selection after transient
transfection" would generate a lot of false positives that many drug
positives won't carry the gene of interest in the genome. It is a real
pain to pick out the right clones. But I also heard that with the new
IRES-containing vectors, the chance to get false positives has
decreased significantly. My cDNA is on a plasmid with Gateway entry
sites. However, as far as I know, Invitrogen doesn't offer an IRES-
based expression vector.

The alternative is to use lentiviral transduction. Most positives are
likely the real ones. However, I was also advised that the expression
level in a lentiviral system is not very high, even with a CMV
promoter, because of the low copy number. Invitrogen does offer a very
attractive product in "ViraPower™ II Lentiviral Lumio™ Gateway
Technology":

https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProductDetails&productDescription=12525&

It is a hard choice to make. I'm new to the mammalian system, any
experienced comments here? Thanks in advance.

Patrick